Rad21l1 cohesin subunit is dispensable for spermatogenesis but not oogenesis in zebrafish

During meiosis I, ring-shaped cohesin complexes play important roles in aiding the proper segregation of homologous chromosomes. RAD21L is a meiosis-specific vertebrate cohesin that is required for spermatogenesis in mice but is dispensable for oogenesis in young animals. The role of this cohesin in other vertebrate models has not been explored. Here, we tested if the zebrafish homolog Rad21l1 is required for meiotic chromosome dynamics during spermatogenesis and oogenesis. We found that Rad21l1 localizes to unsynapsed chromosome axes. It is also found between the axes of the mature tripartite synaptonemal complex (SC) in both sexes. We knocked out rad21l1 and found that nearly all rad21l1^-/- mutants develop as fertile males, suggesting that the mutation causes a defect in juvenile oogenesis, since insufficient oocyte production triggers female to male sex reversal in zebrafish. Sex reversal was partially suppressed by mutation of the checkpoint gene tp53, suggesting that the rad21l1 mutation activates Tp53-mediated apoptosis or arrest in females. This response, however, is not linked to a defect in repairing Spo11-induced double-strand breaks since deletion of spo11 does not suppress the sex reversal phenotype. Compared to tp53 single mutant controls, rad21l1^-/- tp53^-/- double mutant females produce poor quality eggs that often die or develop into malformed embryos. Overall, these results indicate that the absence of rad21l1^-/- females is due to a checkpoint-mediated response and highlight a role for a meiotic-specific cohesin subunit in oogenesis but not spermatogenesis.     

Increased female fertility in aquaporin 8-deficient mice

Aquaporin-8 (AQP8) is a water channel expressed extensively in male and female reproductive systems. But its physiological functions are largely unknown. In the present study, we first found significantly increased number of offspring delivered by AQP8(-/-) mothers compared with wild-type mothers in cross-mating experiments. Comparison of ovulation in the two genotypes demonstrated that AQP8(-/-) ovaries released more oocytes (9.5 ± 1.9 vs. 7.1 ± 2.1 in normal ovulation and 37.8 ± 6.7 vs. 27.9 ± 5.7 in superovulation). Histological analysis showed increased number of corpus luteums in mature AQP8(-/-) ovaries, suggesting increased maturation and ovulation of follicles. By RT-PCR, western blot and immunohistochemistry analyses, we determined the expression of AQP8 in mouse ovarian granulosa cells. Granulosa cells isolated from AQP8(-/-) mice showed 45% of decreased membrane water permeability than wild-type mice. As the atresia of ovarian follicles is primarily due to apoptosis of granulosa cells, we analyzed the apoptosis of isolated granulosa cells from wild-type and AQP8(-/-) mice. The results indicated significantly lower apoptosis rate in AQP8(-/-) granulosa cells (21.3 ± 3.6% vs. 32.6 ± 4.3% in AQP8(+/+) granulosa cells). Taken together, we conclude that AQP8 deficiency increases the number of mature follicles by reducing the apoptosis of granulosa cells, thus increasing the fertility of female mice. This discovery may offer new insight of improving female fertility by reducing granulosa cell apoptosis through AQP8 inhibition.     

Protection from fluorescein isothiocyanate-induced fibrosis in IL-13-deficient, but not IL-4-deficient, mice results from impaired collagen synthesis by fibroblasts

Intratracheal injection of FITC results in acute lung injury and progresses to fibrosis by day 21 postchallenge. In response to FITC, BALB/c mice produce IL-4 and IL-13 in the lung. To investigate whether IL-4 and/or IL-13 were important profibrotic mediators in this model, we examined the fibrotic response to FITC in mice that were genetically deficient in IL-4 (IL-4(-/-)), IL-13 (IL-13(-/-)), or IL-4 and IL-13 combined (IL-4/13(-/-)). Baseline levels of collagen were similar in all mice. In response to FITC, both BALB/c and IL-4(-/-) mice developed fibrosis, whereas the IL-13(-/-) and IL-4/13(-/-) mice were significantly protected, as measured by total lung collagen levels and histology. Total leukocyte recruitment to the lung was similar in all four strains of mice when measured on days 7, 14, and 21 post-FITC. BALB/c mice showed prominent eosinophilia on day 7 that was absent in IL-4(-/-), IL-13(-/-), and IL-4/13(-/-) mice, suggesting that eosinophilia is not necessary for development of a fibrotic response. There were no significant differences in the percentages of any other leukocytes analyzed between the genotypes. Similarly, protection in IL-13(-/-) mice was not associated with alterations in cytokine or eicosanoid profiles. Interestingly, TGF-beta1 production was not reduced in IL-13(-/-) mice. Analyses of fibroblasts isolated from the four genotypes demonstrated that although there were similar numbers of fibroblasts present in cultures of lung minces, fibroblasts from IL-13-deficient strains have reduced basal and stimulated levels of collagen production. IL-13Ralpha1 expression increases on fibroblasts during fibrotic responses in vivo, and IL-13 increases collagen synthesis in fibroblasts. Thus, IL-13 mediates its profibrotic actions through direct effects on fibroblast production of extracellular matrix.     

ARHGEF15 is expressed in undifferentiated spermatogonia but is not required for spermatogenesis in mice

Spermatogenesis is a continual process that relies on the activities of undifferentiated spermatogonia, which contain spermatogonial stem cells (SSCs) that serve as the basis of spermatogenesis. The gene expression pattern and molecular control of fate decisions of undifferentiated spermatogonia are not well understood. Rho guanine nucleotide exchange factor 15 (ARHGEF15, also known as EPHEXIN5) is a guanine nucleotide-exchange factor (GEF) that activates the Rho protein. Here, we reported that ARHGEF15 was expressed in undifferentiated spermatogonia and spermatocytes in mouse testes; however, its deletion did not affect spermatogenesis. Arhgef15^-/- mice were fertile, and histological examination of the seminiferous tubules of Arhgef15^-/- mice revealed complete spermatogenesis with the presence of all types of spermatogenic cells. Proliferation and differentiation of the undifferentiated spermatogonia were not impacted; however, further analysis showed that Arhgef15 deletion resulted in decreased expression of Nanos2, Lin28a and Ddx4. Together, these findings suggest that ARHGEF15 was specifically enriched in undifferentiated spermatogonia and regulated gene expression but dispensable for spermatogenesis in mice.     

The essential roles of Mps1 in spermatogenesis and fertility in mice

Monopolar spindle 1 (MPS1), which plays a critical role in somatic mitosis, has also been revealed to be essential for meiosis I in oocytes. Spermatogenesis is an important process involving successive mitosis and meiosis, but the function of MPS1 in spermatogenesis remains unclear. Here, we generated Mps1 conditional knockout mice and found that Ddx4-cre-driven loss of Mps1 in male mice resulted in depletion of undifferentiated spermatogonial cells and subsequently of differentiated spermatogonia and spermatocytes. In addition, Stra8-cre-driven ablation of Mps1 in male mice led to germ cell loss and fertility reduction. Spermatocytes lacking Mps1 have blocked at the zygotene-to-pachytene transition in the prophase of meiosis I, which may be due to decreased H2B ubiquitination level mediated by MDM2. And the expression of many meiotic genes was decreased, while that of apoptotic genes was increased. Moreover, we also detected increased apoptosis in spermatocytes with Mps1 knockout, which may have been the reason why germ cells were lost. Taken together, our findings indicate that MPS1 is required for mitosis of gonocytes and spermatogonia, differentiation of undifferentiated spermatogonia, and progression of meiosis I in spermatocytes.Subject terms: Cell division, Spermatogenesis     

Loss of gremlin delays primordial follicle assembly but does not affect female fertility in mice

The transforming growth factor beta (TGFB) protein family is renowned for its diverse roles in developmental biology including reproduction. Gremlin is a member of the differential screening-selected gene aberrative in neuroblastoma (DAN)/cerberus family of bone morphogenetic protein (BMP) antagonists. Recent studies on gremlin focus on its involvement in embryonic skeletal, lung, and kidney development. To define the role of gremlin (Grem1) in female reproduction, we analyzed postnatal folliculogenesis using global and conditional knockout (cKO) mice for gremlin. Grem1(-/-) mice die within 48 h after birth, and ovaries collected from neonatal Grem1(-/-) mice demonstrated reduced oocyte numbers and delayed primordial follicle development. Transplanting Grem1(-/-) neonatal ovaries showed that folliculogenesis proceeded to large antral follicle stage, but Grem1(-/-) ovaries contained corpora lutea-like structures not found in control-transplanted ovaries. However, Grem1 cKO mice had comparable fertility to control mice. These data suggest that gremlin plays a previously uncharacterized role in the regulation of oocyte numbers and the timing of primordial follicle development, but either it is not required for later folliculogenesis or its loss is possibly compensated by other BMP antagonists.     

Thymic alterations in EphA4-deficient mice

In the present work, we have demonstrated in vivo an altered maturation of the thymic epithelium that results in defective T cell development which increases with age, in the thymus of Eph A4-deficient mice. The deficient thymi are hypocellular and show decreased proportions of double-positive (CD4+CD8+) cells which reach minimal numbers in 4-wk-old thymi. The EphA4 (-/-) phenotype correlates with an early block of T cell precursor differentiation that results in accumulation of CD44-CD25+ triple-negative cells and, sometimes, of CD44+CD25- triple-negative thymocytes as well as with increased numbers of apoptotic cells and an important reduction in the numbers of cycling thymocytes. Various approaches support a key role of the thymic epithelial cells in the observed phenotype. Thymic cytoarchitecture undergoes profound changes earlier than those found in the thymocyte maturation. Thymic cortex is extremely reduced and consists of densely packed thymic epithelial cells. Presumably the lack of forward Eph A4 signaling in the Eph A4 -/- epithelial cells affects their development and finally results in altered T cell development.     

Smcy transgene does not rescue spermatogenesis in sex-reversed mice

In mouse, the Sxr^b deletion interval (delta Sxr^b) maps to the small short arm of the Y chromosome and is known to contain gene(s) required for normal spermatogenesis; in particular, Spy, which is essential for the postnatal mitotic proliferation of spermatogonia. This deletion interval is approximately 1–2 Mb and contains eight known genes. In this paper we report the construction of YAC transgenic mice containing different regions of the delta Sxr^b interval including Zfy1, Ube1y, Smcy, and Eif2s3. Two male and one female founder mice, transgenic for all four genes, were sterile. However, a fertile transgenic, carrying a full-length copy of the Smcy gene integrated into central Chr 12, was identified. Smcy is a highly conserved Y chromosome-located gene, encoding peptides corresponding to epitopes of the male-specific antigen, H-Y. The Smcy transgene was ubiquitously expressed in all organs and tissues tested in male and female carriers. Introduction of the transgene into an X Sxr^b/O genetic background did not rescue the early arrest of spermatogenesis characteristic of these males. These data indicate that the presence of Smcy is not sufficient to restore spermatogenesis, making it a highly unlikely candidate for Spy. Received: 16 June 2000 / Accepted: 25 September 2000     

Reduced fertility and spermatogenesis defects in mice lacking chromosomal protein Hmgb2

High mobility group 2 protein (Hmgb2) is a member of the HMGB protein family, which includes the ubiquitous Hmgb1 and the embryo-specific Hmgb3. The three proteins are more than 80% identical at the amino acid level and their biochemical properties are indistinguishable. Hmgb1 is an abundant component of all mammalian nuclei and acts as an architectural factor that bends DNA and promotes protein assembly on specific DNA targets. Cells that lack Hmgb1 can survive, although mutant mice die shortly after birth. As Hmgb2 is present in all cultured cells and is abundant in thymus, the preferred source for HMGB proteins, it was considered a ubiquitous variant of Hmgb1. We show that in adult mice Hmgb2 is restricted mainly to lymphoid organs and testes, although it is widely expressed during embryogenesis. Mice that lack Hmgb2 are viable. However, male Hmgb2(-/-) mice have reduced fertility, that correlates with Sertoli and germ cell degeneration in seminiferous tubules and immotile spermatozoa. Significantly, Hmgb2 is expressed at very high levels in primary spermatocytes, while it is barely detectable in spermatogonia and elongated spermatids. This peculiar pattern of expression and the phenotype of mutants indicate that Hmgb2 has a specialised role in germ cell differentiation.     

Mice lacking the UBC4-testis gene have a delay in postnatal testis development but normal spermatogenesis and fertility

Activation of ubiquitination occurs during spermatogenesis and is dependent on the induction of isoforms of the UBC4 family of ubiquitin-conjugating enzymes. The UBC4-testis isoform is testis specific, is induced in round spermatids, and demonstrates biochemical functions distinct from a ubiquitously expressed isoform UBC4-1. To explore further the function of UBC4-testis, mice bearing inactivation of this gene were produced. Homozygous (-/-) mice showed normal body growth and fertility. Although testis weight and morphology were normal in testes from adult mice, examination of young mice during the first wave of spermatogenesis revealed that testes were approximately 10% smaller in weight at 40 and 45 days of age but had become normal at 65 days of age. Overall protein content, levels of ubiquitinated proteins, and ubiquitin-conjugating activity did not differ between wild-type and homozygous (-/-) mice. Spermatid number, as well as the motility of spermatozoa isolated from the epididymis, was also normal in homozygous (-/-) mice. To determine whether the germ cells lacking UBC4-testis might be more sensitive to stress, testes from wild-type and knockout mice were exposed to heat stress by implantation in the abdominal cavity. Testes from both strains of mice showed similar rates of degeneration in response to heat. The lack of an obvious phenotype did not appear to be due to induction of other UBC4 isoforms, as shown by two-dimensional gel immunoblotting. Our data indicate that UBC4-testis plays a role in early maturation of the testis and suggest that the many UBC4 isoforms have mixed redundant and specific functions.     

Melatonin ameliorates paclitaxel‐induced mice spermatogenesis and fertility defects

Chemotherapeutic drug of paclitaxel (PTX) has been shown to cause reproductive toxicity thus affecting male fertility, but its underlying molecular basis is unclear. Melatonin (MLT) can mitigate the reproductive damage caused by certain chemotherapy drugs. In this study, we aimed to identify impact of PTX on the main biological processes and protective effect of MLT on reproductive damage caused by PTX. Mice exposed to PTX mainly impaired spermatogenesis, such as decreased sperm counts, reduced sperm motility and increased abnormal sperm. Decreased expressions of germ cell proliferation‐associated protein PCNA and meiosis‐related protein SYCP3 induced by PTX were determined by Western blot, while MLT ameliorated this effect and increased the expressions of PCNA, SYCP3, DMC1, STRA8 and fertility‐related protein of HSPA2, resulting in significantly improved spermatogenesis and sperm quality levels. In vitro fertilization experiment showed that PTX significantly decreased blastocyst formation rates, which can be improved by MLT administration, but not two‐cell development rates. Taken together, this work demonstrated PTX can adversely affect germ cell proliferation and meiosis, which ultimately influence sperm quality and male fertility, and highlighted the protective ability of MLT on ameliorating the side effects of PTX, especially on sperm quality. The results provide information to further the study on the molecular mechanism of PTX''s effects on male reproduction and the protective mechanism of MLT.     

Cul4A is essential for spermatogenesis and male fertility

The mammalian Cul4 genes, Cul4A and Cul4B, encode the scaffold components of the cullin-based E3 ubiquitin ligases. The two Cul4 genes are functionally redundant. Recent study indicated that mice expressing a truncated CUL4A that fails to interact with its functional partner ROC1 exhibit no developmental phenotype. We generated a Cul4A−/− strain lacking exons 4–8 that does not express any detectable truncated protein. In this strain, the male mice are infertile and exhibit severe deficiencies in spermatogenesis. The primary spermatocytes are deficient in progression through late prophase I, a time point when expression of the X-linked Cul4B gene is silenced due to meiotic sex chromosome inactivation. Testes of the Cul4A−/− mice exhibit extensive apoptosis. Interestingly, the pachytene spermatocytes exhibit persistent double stranded breaks, suggesting a deficiency in homologous recombination. Also, we find that CUL4A localizes to the double stranded breaks generated in pre-pachytene spermatocytes. The observations identify a novel function of CUL4A in meiotic recombination and demonstrate an essential role of CUL4A in spermatogenesis.     

Tumor necrosis factor alpha-deficient, but not interleukin-6-deficient, mice resist peripheral infection with scrapie

In most peripheral infections of rodents and sheep with scrapie, infectivity is found first in lymphoid tissues and later in the central nervous system (CNS). Cells within the germinal centers (GCs) of the spleen and lymph nodes are important sites of extraneural replication, from which infection is likely to spread to the CNS along peripheral nerves. Here, using immunodeficient mice, we investigate the identity of the cells in the spleen that are important for disease propagation. Despite possessing functional T and B lymphocytes, tumor necrosis factor alpha-deficient (TNF-alpha(-/-)) mice lack GCs and follicular dendritic cell (FDC) networks in lymphoid tissues. In contrast, lymphoid tissues of interleukin-6-deficient (IL-6(-/-)) mice possess FDC networks but have impaired GCs. When the CNSs of TNF-alpha(-/-), IL-6(-/-), and wild-type mice were directly challenged with the ME7 scrapie strain, 100% of the mice were susceptible, developing disease after closely similar incubation periods. However, when challenged peripherally (intraperitoneally), most TNF-alpha(-/-) mice failed to develop scrapie up to 503 days postinjection. All wild-type and IL-6(-/-) mice succumbed to disease approximately 300 days after the peripheral challenge. High levels of scrapie infection and the disease-specific isomer of the prion protein, PrP(Sc), were detectable in spleens from challenged wild-type and IL-6(-/-) mice but not from TNF-alpha(-/-) mice. Histopathological analysis of spleen tissue demonstrated heavy PrP accumulations in direct association with FDCs in challenged wild-type and IL-6(-/-) mice. No PrP(Sc) accumulation was detected in spleens from TNF-alpha(-/-) mice. We conclude that, for the ME7 scrapie strain, mature FDCs are critical for replication in lymphoid tissues and that in their absence, neuroinvasion following peripheral challenge is impaired.     

Interplay between p53 and Ink4c in spermatogenesis and fertility

The tumor suppressor p53, and the cyclin-dependent kinase inhibitor Ink4c, have been both implicated in spermatogenesis control. Both p53-/- and Ink4c-/- single knockout male mice are fertile, despite testicular hypertrophy, Leydig cell differentiation defect, and increased sperm count in Ink4c-/- males. To investigate their collaborative roles, we studied p53-/- Ink4c-/- dual knockout animals, and found that male p53-/- Ink4c-/- mice have profoundly reduced fertility. Dual knockout male mice show a marked decrease in sperm count, abnormal sperm morphology and motility, prolongation of spermatozoa proliferation and delay of meiosis entry, and accumulation of DNA damage. Genetic studies showed that the effects of p53 loss on fertility are independent of its downstream effector Cdkn1a. Absence of p53 also partially reverses the hyperplasia seen upon Ink4c loss, and normalizes the Leydig cell differentiation defect. These results implicate p53 in mitigating both the delayed entry into meiosis and the secondary apoptotic response that occur in the absence of Ink4c. We conclude that the cell cycle genes p53 and Ink4c collaborate in sperm cell development and differentiation, and may be important candidates to investigate in human male infertility conditions.     

Late onset of spermatogenesis and gain of fertility in POG-deficient mice indicate that POG is not necessary for the proliferation of spermatogonia

The germ cell-deficient (gcd) mouse mutation is a recessive, transgenic insertional mutation associated with the disruption of two Chr11 genes, Pog (proliferation of germ cells) and Vrk2 (vaccinia virus-related protein kinase 2). We have recently shown that like gcd/gcd mice, targeted Pog-/- males and females show virtually no spermatogenesis or oogenesis at 4-6 wk of age. Because Pog is deleted in gcd/gcd and Pog-/- mice, a comparison of the phenotypes of the two mouse models is appropriate. Here, we report that unlike in POG-deficient females, the germ cells in POG-deficient males eventually populate the seminiferous tubules at 9 wk, and fertility can be achieved by 12 wk. Homozygous gcd/gcd males did not show a similar degree of germ cell population, and most gcd/gcd males remained infertile at 16 and 22 wk of age. A comparison of the degree of germ cell deficiency at 13.5 days postcoitum and 1 day postpartum between Pog-/- and gcd/gcd males revealed that gcd/gcd males had far fewer germ cells than Pog-/- males at both time points. Our data suggest that Pog is essential for proper primordial germ cell proliferation in the embryonic stage but is not needed for spermatogonial proliferation after birth. Thus, the difference in the spermatogenetic potential in adult Pog-/- and gcd/gcd mice may result from the severity of germ cell deficiency rather than from the inability of gcd/gcd spermatogonia to proliferate efficiently. The greater deficiency of germ cells before the onset of spermatogenesis seen in gcd/gcd males compared to Pog-/- mice suggests either that the different background affects the outcome of Pog deletion or that Vrk2 has additional effects on germ cell development.     

Reduction of copper and metallothionein in toxic milk mice by tetrathiomolybdate, but not deferiprone.

Copper is both essential for life and toxic. Aberrant regulation of copper at the level of intracellular transport has been associated with inherited diseases, including Wilson's disease (WND) in humans. WND results in accumulation of copper and the copper and zinc-binding protein metallothionein (MT) in liver and other tissues, liver degeneration, and neurological dysfunction. The toxic milk (TX) mutation in mice results in a phenotype that mimics human WND, and TX has been proposed to be a model of the disease. We characterized TX mice as a model of altered metal ion and MT levels during development, and after treatment with the metal ion chelators tetrathiomolybdate (TTM) and deferiprone (L1). We report that hepatic, renal and brain copper and MT are elevated in TX mice at 3 and 12 months of age. Zinc was significantly higher in TX mouse liver, but not brain and kidney, at both time points. Nodules appeared spontaneously in TX mouse livers at 8-12 months that maintained high copper levels, but with more normal morphology and decreased MT levels. Treatment of TX mice with TTM significantly reduced elevated hepatic copper and MT. Transient increases in blood and kidney copper accompanied TTM treatment and indicated that renal excretion was a significant route of removal. Treatment with L1, on the other hand, had no effect on liver or kidney copper and MT, but resulted in increased brain copper and MT levels. These data indicate that TTM, but not L1, may be useful in treating diseases of copper overload including WND.